Mucosal sites are continuously exposed to pathogenic microorganisms and are therefore equipped to control respiratory infections. Type 3 innate lymphoid cells (ILC3) are key players in antimicrobial defense in intestinal mucosa, through interleukin 17 and interleukin 22 (IL-22) production. The present study aimed at analyzing the distribution and function of ILC3 in the respiratory tract. We first observed that lung mucosa harbors a discrete population of ILC3 expressing CD127, CD90, CCR6, and the transcriptional factor RORγt. In addition, lung ILC3 were identified as a major source of IL-22 in response to interleukin 23 stimulation. During Streptococcus pneumoniae infection, ILC3 rapidly accumulated in the lung tissue to produce IL-22. In response to S. pneumoniae, dendritic cells and MyD88, an important adaptor of innate immunity, play critical functions in IL-22 production by ILC3. Finally, administration of the Toll-like receptor 5 agonist flagellin during S. pneumoniae challenge exacerbated IL-22 production by ILC3, a process that protects against lethal infection. In conclusion, boosting lung ILC3 might represent an interesting strategy to fight respiratory bacterial infections.

In E. coli, 10 to 15% of growing bacteria produce dimeric chromosomes during DNA replication. These dimers are resolved by XerC and XerD, two tyrosine recombinases that target the 28-nucleotide motif (dif) associated with the chromosome's replication terminus. In streptococci and lactococci, an alternative system is composed of a unique, Xer-like recombinase (XerS) genetically linked to a dif-like motif (dif SL) located at the replication terminus. Preliminary observations have suggested that the dif/Xer system is commonly found in bacteria with circular chromosomes but that assumption has not been confirmed in an exhaustive analysis. The aim of the present study was to extensively characterize the dif/Xer system in the proteobacteria, since this taxon accounts for the majority of genomes sequenced to date. To that end, we analyzed 234 chromosomes from 156 proteobacterial species and showed that most species (87.8%) harbor XerC and XerD-like recombinases and a dif-related sequence which (i) is located in non-coding sequences, (ii) is close to the replication terminus (as defined by the cumulative GC skew) (iii) has a palindromic structure, (iv) is encoded by a low G+C content and (v) contains a highly conserved XerD binding site. However, not all proteobacteria display this dif/XerCD system. Indeed, a sub-group of pathogenic ε-proteobacteria (including Helicobacter sp and Campylobacter sp) harbors a different recombination system, composed of a single recombinase (XerH) which is phylogenetically distinct from the other Xer recombinases and a motif (dif H) sharing homologies with dif SL. Furthermore, no homologs to dif or Xer recombinases could be detected in small endosymbiont genomes or in certain bacteria with larger chromosomes like the Legionellales. This raises the question of the presence of other chromosomal deconcatenation systems in these species. Our study highlights the complexity of dif/Xer recombinase systems in proteobacteria and paves the way for systematic detection of these components in prokaryotes.

Material Supplementary 5.DC1http://www.jimmunol.org/content/suppl/2010/06/21/jimmunol.100011

The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush borderassociated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

SummaryThe fimbrial and afimbrial adhesins of the Dr family mediate the adherence of uropathogenic and diarrhoea-associated Escherichia coli to decay-accelerating factor (DAF) present on erythrocytes and other cell types. The Dr haemagglutinin binds type IV collagen and, unlike other members of the Dr family, mediates an adherence inhibited in the presence of chloramphenicol. We examined the ability of other members of the Dr family-AFAI, AFAIII, and F1845-to bind to type IV collagen, and demonstrated that the collagen-binding phenotype was unique to the Dr haemagglutinin. We employed site-directed mutagenesis to demonstrate the requirement of a negatively charged amino-acid at position 54 of the Dr haemagglutinin subunit for chloramphenicol sensitivity of binding. Mutations at position 32, 40, 54, 90, and 113 differently affected type IV collagen binding and chloramphenicol sensitivity of binding, while retaining DAF-binding capability. These results suggest the existence of a conformational receptor-binding domain in the major structural subunit of Dr family adhesins and demonstrate that chloramphenicol sensitivity of binding and adherence to type IV collagen were independent and separable phenotypes. Finally, we showed that the two conserved cysteine residues of Dr family structural subunits form a disulphide bond and that mutations of these residues abolish haemagglutination and binding to type IV collagen.

Diffusely adhering Escherichia coli (DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. Using draE mutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.Diffusely adhering Escherichia coli (DAEC) strains are considered a heterogeneous group. It has been well established that some Afa/Dr DAEC expressing related adhesins adhere to host cells and cause symptomatic urinary tract and intestinal infections. Afa/Dr DAEC harboring the afimbrial adhesin I (AfaE-I) (34) and adhesin III (AfaE-III) (18, 35), the Dr hemagglutinin (51), and the adhesin DR-II (58) have been associated with 30% of cases of pyelonephritis in pregnant women. Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin has been isolated from an infant with diarrhea (4). These virulent E. coli strains express a family of gene operons, including afa (19,20,30,34,35), dra (66), and daa (3, 4, 38). Moreover, a common system of adhesion involving the decay-accelerating-factor (DAF; CD55) as a receptor has been identified for Afa/Dr DAEC (48, 50).Yamamoto et al. (67) were first to report that adherence of DAEC induced elongated cellular projections in epithelial HeLa cells. Cookson and Nataro (10) observed that attachment of Afa/Dr DAEC onto epithelial Hep-2 cells is followed by induction of a long thin membrane extending from the cell surface. Moreover, Afa/Dr DAEC-induced cytoskeletal rearrangements in HeLa cells have been recently reported by Goluszko et al. (23). We have previously shown that Afa/Dr DAEC strains infect polarized human intestinal Caco-2 cells expressing a well-characterized brush border endowed with CD55 and forming a monolayer mimicking an epithelial barrier (32). When investi...

Motility is often a pathogenicity determinant of bacteria targeting mucosal tissues. Flagella constitute the machinery that propels bacteria into appropriate niches. Besides motility, the structural component, flagellin, which forms the flagella, targets Toll-like receptor 5 (TLR5) to activate innate immunity. The compartmentalization of flagellin-mediated immunity and the contribution of epithelial cells and dendritic cells in detecting flagellin within luminal and basal sides are highlighted here, respectively. While a direct stimulation of the epithelium mainly results in recruitment of immune cells and production of antimicrobial molecules, TLR5 engagement on parenchymal dendritic cells can contribute to the stimulation of innate lymphocytes such as type 3 innate lymphoid cells, as well as T helper cells. This review, therefore, illustrates how the innate and adaptive immunity to flagellin are differentially regulated by the epithelium and the dendritic cells in response to pathogens that either colonize or invade mucosa.

The adhesion of Pseudomonas aeruginosa to type 1 (Gal,11-3GlcNAc) and type 2 (Galpll-4GlcNAc) disaccharide determinants was studied in a microtiter adhesion assay and a thin-layer chromatography bacterial overlay assay. The oligosaccharides were prepared from human breast milk and human urine and were conjugated to hexadecylaniline to form neoglycolipids that were used in the assays. Both the mucoid and the nonmucoid strains that were studied recognized the disaccharide determinants. Sialylation of the oligosaccharides did not suppress binding in the thin-layer chromatography assay, but t2-6-linked sialic acid blocked binding in the microtiter assay. The use of bovine serum albumin instead of gelatin as a blocking agent against nonspecific binding completely suppressed binding in the thin-layer chromatography assay. Isogenic nonpiliated mutants of nonmucoid strains constructed by interrupting the pilin gene retained their adhesive capacity for the disaccharide units, indicating that binding to the disaccharides was mediated by a nonpilus adhesin(s). Furthermore, two monoclonal antibodies that recognize the type 2 disaccharide determinant (GaI01-4GlcNAc) partially inhibited adhesion of a pair of piliated and nonpiliated isogenic strains to mucin. This study suggests that P. aeruginosa utilizes a nonpilus adhesin(s) to bind to disaccharide units commonly found in mucins, in addition to pili and alginate, two previously described adhesins.